Application of DNA Quadruplex Hydrogels Prepared from Polyethylene Glycol-Oligodeoxynucleotide Conjugates to Cell Culture Media.

Akinori Kuzuya,Shizuma Tanaka,Yuhei Hachiro,Shinsuke Yukami,Yuichi Ohya

doi:10.3390/polym11101607

Abstract

Application of Na+-responsive DNA quadruplex hydrogels, which utilize G-quadruplexes as crosslinking points of poly(ethylene glycol) (PEG) network as cell culture substrate, has been examined. PEG-oligodeoxynucleotide (ODN) conjugate, in which four deoxyguanosine (dG4) residues are tethered to both ends of PEG, was prepared by modified high-efficiency liquid phase (HELP) synthesis of oligonucleotides and used as the macromonomer. When mixed with equal volume of cell culture media, the solution of PEG-ODN turned into stiff hydrogel (G-quadruplex hydrogel) as the result of G-quadruplex formation by the dG4 segments in the presence of Na+. PEG-ODN itself did not show cytotoxicity and the resulting hydrogel was stable enough under cell culture conditions. However, L929 fibroblast cells cultured in G-quadruplex hydrogel remained spherical for a week, yet alive, without proliferation. The cells gradually sedimented through the gel day by day, probably due to the reversible nature of G-quadruplex formation and the resulting slow rearrangement of the macromonomers. Once they reached the bottom glass surface, the cells started to spread and proliferate.

Highlights

Recent development of various hydrogels with unique properties is attracting scientists from material chemistry, and from broad research areas such as medical science to the field [1,2]
We have recently developed a new class of hydrogels utilizing such DNA quadruplexes as cross-linking points of the 3D polymer network [25,26,27]
Polyethylene glycol-oligodeoxynucleotide (PEG-ODN) conjugates bearing merely four deoxyguanosine residues for G-quadruplexes or five deoxycytidine residues for i-motifs at the ends of linear or four-way branched poly(ethylene glycol) (PEG), were prepared as macromonomers by applying high-efficiency liquid phase (HELP) synthesis of oligonucleotides developed by Bonora et al with a few modifications [28]

Summary

Introduction

Recent development of various hydrogels with unique properties is attracting scientists from material chemistry, and from broad research areas such as medical science to the field [1,2]. Polyethylene glycol-oligodeoxynucleotide (PEG-ODN) conjugates bearing merely four deoxyguanosine residues (dG4) for G-quadruplexes or five deoxycytidine residues (dC5) for i-motifs at the ends of linear or four-way branched PEG, were prepared as macromonomers by applying high-efficiency liquid phase (HELP) synthesis of oligonucleotides developed by Bonora et al with a few modifications [28]. This technique enabled us to prepare typically 10−20 g of PEG-ODN conjugates in laboratories and to overcome the size-barrier of popular solid-phase DNA synthesis [29], which only produces

Methods

Results

Conclusion